Summary Background hsp27 is a member of the small heat shock protein family. Its expression in epidermal keratinocytes in situ and in tissue culture correlates with differentiation. Experimental evidence points to the fact that hsp27 is a molecular chaperone and is involved in the regulation of cell growth and differentiation. Objectives To investigate whether epidermal hsp27 through its chaperone function plays a role in the assembly of keratin filaments and the cornified cell envelope. Methods We performed double staining immunofluorescence and immunogold microscopy on normal human skin (n = 15). We analysed the colocalization of hsp27 with actin, keratins and proteins of the cornified cell envelope (loricrin, filaggrin, transglutaminase 1). Results Actin staining did not reveal detectable colocalization with hsp27. For keratins, transglutaminase, loricrin and filaggrin colocalization was found in more than 60% of the samples. Colocalization was confined to a narrow subcorneal layer with varying patterns of expression. Electron microscopy revealed that loricrin and filaggrin colocalize with hsp27 indirectly through binding to intermediate filaments. Conclusions These results provide morphological evidence that in normal human skin hsp27 might act as a chaperone of cornification. Investigations of the molecular hsp27 interactions with the proteins of the cornified cell envelope are necessary to gain further insight into terminal keratinocyte differentiation and disorders of keratinization.